We have shown the presence of angiotensin (Ang) H receptors in the myocardium, sympathetic nerves and conduction system of the human heart. Ang II elicits a positive inotropic effect in the human heart. In homogenates of the human cardiac ventricle, the major (>75%) enzymatic conversion of Ang I to Ang II is produced by a serine proteinase that is not inhibited by Ang I-converting enzyme (ACE) inhibitors. We have purified and cloned this proteinase. Structurally, this enzyme belongs to the chymase group of chymotrypsin-like proteinases and will be referred to as human heart chymase (hHC). Unlike chymotrypsin and, indeed, other chymases, hHC is the most specific and efficient Ang II- forming enzyme described. Preliminary studies using cardiac trabeculae show the presence of a functional pathway for Ang II formation in the human heart independent of the ACE pathway. Because high levels of immunoreactive-hHC are associated with the extracellular matrix surrounding myocytes, we speculate that the ACE-independent pathway of cardiac Ang II formation is indeed hHC-dependent. We propose to develop specific substrate-and antibody-based inhibitors of hHC to definitively establish that hHC is the major functional Ang II- forming enzyme in the human heart. These inhibitors will be used to show that the major conversion of Ang I to Ang II in human cardiac trabeculae, and that the response elicited by Ang I, are due to hHC. In parallel studies, we plan to crystallize hHC in the native form and in the inhibitor complexed form to study the unique structural determinants of hHC responsible for its high substrate specifity. Studies are also planned to functionally map the extended substrate binding site of hHC using Ang I analogs. In the human heart, immunoreactive-hHC is stored in cardiac mast cell granules. To study the processing of hHC, we propose to develop a human cardiac mast cell line that expresses hHC. This cell line will also be used to study the cis-acting elements and trans-acting factors that allow only a subset of mast cells to express hHC. The presence of hHC in the human heart suggests that cardiac Ang II formation may not cease during the treatment of hypertension and congestive heart failure with ACE inhibitors. The achievement of the goals of the proposed studies will ultimately help us understand the consequence of cardiac Ang II formation in the normal and failing heart, and will improve our understanding of heart failure and its treatment.